RESEARCH PLAN: TISSUE PROCESSING AND CONFOCAL MICROSCOPY MODULE OVERVIEW This module will meet the demands of several NEI funded researchers who require assistance with tissue processing for histology, immunohistology, and confocal microscopy. Many investigators in the Ophthalmology department have multiple animal studies which require rapid characterization of eye tissues in perspective of histology, immunohistochemistry, and light microscopy as well as confocal microscopy. For these purposes, cryosectioning, paraffin sectioning, and flat amount technique are needed. Eye tissues are unique because eye wall is comprised of several layers which are easy to separate during histological processing and the separation will adversely affect scientific evaluation of morphology and anatomical relationship of the study interests. Therefore, sending out small ocular tissue samples for histology outside the vision research community is not only costly and ineffective but results in suboptimal sections and evaluations because non-vision oriented laboratories are not experienced in the techniques required to properly process ocular tissues. Another important consideration is to accurately localize the study area and do the focal processing for histology evaluation. To accurately localize the study interests which often times are in millimeter or micrometer scales require knowledge of eye anatomy and familiarity of clinical ophthalmic instrument such as indirect ophthalmoscope and slit-lamp. The ability to have these services performed by a core laboratory located in an ophthalmology department will yield more reliable results from technicians who have an understanding of vision research. In addition, evaluation using light and confocal microscopy with an expert who is experienced with eye pathology and eye diseases is also a needed service for the other vision investigators. The resources and the services will be centralized in new space assigned to the Tissue Processing and Confocal Microscopy module in Shiley Eye Center SEC Room 210 (298 sq ft). The dedication of new lab space for this module has several advantages including: 1) the histotechnician can service both the tissue processing and confocal microscopy as the two laboratories are on the same floor and 2) the tissue fixation, processing and staining and confocal microscopy will be in direct proximity to the expert faculty (Cheng, Freeman and Silva) as well as to many of the users. Confocal Imaging.- The Jacobs Retina Center (room 229, 448 sq ft) in the Department of Ophthalmology houses one confocal system an Olympus 1X81 inverted fluorescence confocal microscope, which uses a Hamamatsu ORCA-ER digital camera for imaging. Upgrades to the confocal microscope with resources form this module are planned with 60x water immersion lens. Confocal maintenance, supervision, training and online scheduling are managed by Dr. Gabriel Silva and his technicians. These facilities have been used in the past by Drs. Cheng, Freeman, Ayyagari, and Silva, and the objective of this module is to make the equipment and services more readily available to all the participating investigators. Currently, the confocal microscope is not used to its capacity; rather it is used approximately 50% of the time. By providing a dedicated histotechnician with imaging training this valuable piece of equipment can be utilized by more vision researchers. Dr. Silva is an expert in confocal imaging and has agreed to make this microscope available to others (see Dr, Silva's letter of support). Confocal microscopy allows analysis of individual cells and carefully prepared tissue samples in a manner very difficult to do with conventional histology. With this system, multiple markers can be used to evaluate cell response and function using the multiple fluorescent channels of the confocal microscope. This will have an important impact on each investigator's research project as outlined below. Tissue Processing: Many UCSD funded eye research grants carry out animal studies, requiring tissue processing and sectioning. Paraffin embedding and sectioning is extensively used by several investigators, and this requires proper tissue orientation and labeling as well as embedding and sectioning. Our objective is to centralize our facilities and make such services and expertise available to all investigators. There is also a need for cryosectioning by several investigators, so that cryosections of unfixed tissue may be obtained with better immunohistology results. Tissue processing and sectioning are currently performed by Drs. Freeman and Cheng's lab staff research associates based on Drs. Freeman and Cheng's research projects' needs. Other investigators (Drs. Ayyagari and Weinreb) currently send out their tissue to other U.S. institutions for sectioning. The current cryostat and tissue processor are old and inefficient. There is a need to upgrade these two pieces of equipment and hire a full-time histotechnician to carry out these duties to accommodate 7 core module investigators' needs. Dr. Cheng is an expert in ocular pathology and will supervise the tissue processing and provide interpreting service for the histology evaluation along with Dr. Freeman, a professor of ophthalmology and a retina specialist. Dr. Freeman is very experienced with the retinal diseases and retinal morphology. Confocal microscopy requires specific expertise in performing staining and tissue preparation as well as scanning and data storage and interpretation. This also will be performed by the histotechnician to be hired with consultation from Gabriel Silva, Ph.D. a Professor in the Department of Ophthalmology and Department of Bioengineering, an expert in confocal microscopy. Dr. Silva's retina research involves retinal cell regenerate and cell-to-cell communication. Dr. Silva has agreed that the confocal microscope will be made available to all module investigators; it is currently used at approximately 50% of its capacity. The tissue processing and confocal microscopy module also requires resources to upgrade the cryostat and tissue processor to accommodate the growing need for a larger volume of tissue processing. With the newer system, the quality of the sample processing will be enhanced greatly and will allow us to do faster and more versatile immunohistochemistry study. This will greatly enhance each investigator's research project progress and research quality. Staining and Immunohistochemistry: All module investigators with non-clinical research utilize histology and immunostaining in their studies. This module will provide a histology service for the investigators, as there is a need for faster turn-around of results and analysis of pathology. The Cheng and Freeman's lab has available the necessary equipment and staining supplies, but there is a need for a histotechnician to perform this service efficiently at a dedicated core lab on a large scale for all investigators.